Various metabolic diseases caused by a high‐fat diet (HFD) are closely related to gut microbiota dysbiosis and epithelial barrier dysfunction. Polycan, a type of β‐glucan, is effective in treating anti‐obesity and metabolic diseases caused by HFD. However, the effect of Polycan on dysbiosis and epithelial barrier damage is still unknown. In this study, the effects of Polycan on dysbiosis and intestinal barrier damage were investigated using HFD‐induced obese model mice. C57BL/6 mice were fed a HFD for 12 weeks and treated with two different doses of Polycan (250 and 500 mg/kg) orally administered during weeks 9 to 12. Polycan supplementation increased the expression of tight junction genes (zonula occludens‐1, occludin, and claudin‐3) and short‐chain fatty acid (SCFA) content while reducing toxic substances (phenol, p‐cresol, and skatole). Most significantly, Polycan enriched SCFA‐producing bacteria (i.e., Phocaeicola, Bacteroides, Faecalibaculum, Oscillibacter, Lachnospiraceae, and Muribaculaceae), and decreased the Firmicutes/Bacteroidetes ratio and toxic substances‐producing bacteria (i.e., Olsenella, Clostridium XVIII, and Schaedlerella). Furthermore, microbial functional capacity prediction of the gut microbiota revealed that Polycan enriched many SCFA‐related KEGG enzymes while toxic substance‐related KEGG enzymes were depleted. These findings indicated that Polycan has the potential to alleviate HFD‐induced intestinal barrier damage by modulating the function and composition of the gut microbiota.